Functional groups of the highly conserved uridine at position 33 in the anticodon loop of yeast tRNAPhe were altered by a synthetic protocol that replaces U-33 with any desired nucleotide and leaves all other nucleotides of the tRNA intact. The U-33-substituted tRNAs were prepared in an eight-step protocol that begins with partial cleavage of tRNAPhe at U-33 by ribonuclease A. By use of the combined half-molecules as substrate, U-33 was removed from the 5' half-molecule in three steps and then replaced by using RNA ligase to add the desired nucleoside 3',5'-bisphosphate. Each position 33 substituted 5' half-molecule was isolated and annealed to the original 3' half-molecule from the ribonucelase A digestion. The two halves were then rejoined in three steps to give a full-size tRNAPhe variant. This protocol should be applicable to other RNA molecules where a nucleotide substitution is desired at the 5' side of an available unique cleavage site. Seven substituted tRNAPhes containing uridine, pseudouridine, 3-methyluridine, 2'-Omethyluridine, cytidine, deoxycytidine, and purine riboside at position 33 were assayed for aminoacylation with yeast phenylalanyl-tRNA synthetase. Each of the seven tRNAs aminoacylated normally. Thus, unlike the adjacent guanine residue at position 34, U-33 is not involved in the interaction between yeast tRNAPhe and yeast phenylalanyl-tRNA synthetase.
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